ABSTRACT Pediatric low-grade astrocytomas (PLGAs) as a group are the most common solid tumor in children. While rarely fatal, patients frequently experience a relapsing/remitting course wherein repeated cycles of chemotherapy or radiation are required to contain the disease, often inducing irreparable neurologic damage. Therefore, less toxic therapies are urgently needed. We and others have identified genetic drivers of PLGAs in order to develop more specific and effective targeted therapeutic strategies. This proposal addresses the second most common set of alterations in PLGAs, those involving MYB transcription factors (TFs). In recent studies we identified rearrangements involving MYB and MYBL1 in 10% of PLGAs. Each rearrangement is associated with distinct PLGA subtypes. Angiocentric gliomas harbor fusions between a truncated MYB and a truncated portion of the tumor suppressor and myelination gene, QKI. A second tumor subtype, diffuse astrocytoma, frequently exhibits MYBL1 truncation as the only oncogenic change. Similar genetic rearrangements of MYB/MYBL1 have now been implicated in leukemias and adenoid cystic carcinomas. While rearrangements in MYB/MYBL1 are common driver events in PLGAs the biological consequences of these oncogenic changes are not understood. To develop new therapeutic approaches we will determine how the distinct mutant MYB transcription factors contribute to oncogenesis, and whether altered QKI function is critical for growth of angiocentric gliomas. We will address the biology of MYB/MYBL1 oncogenes and implications for new therapies in the following Specific Aims: Aim 1: Test the hypothesis that MYB and MYBL1 alterations contribute to tumorigenesis via distinct but related mechanisms using ChIP-Seq and RNA-Seq to define transcription factor and signaling networks activated by MYB genes. Aim 2: Test the hypothesis that the MYB fusion partner QKI contributes to tumorigenesis by altering RNA processing. We will determine whether MYB- QKI retains RNA binding ability and thereby alters RNA splicing. Aim 3: Identify MYB activated genes functionally critical for tumor growth and amenable to targeting with small molecule therapeutics. This will be done by evaluating novel therapeutics to target MYB/MYBL1 protein regulation and using a CRISPR-screen to identify key gene dependencies for MYB-induced tumors. These studies will provide basic biological insights into the function of MYB proteins and QKI in pediatric astrocytomas and other cancers with MYB alterations. This knowledge will directly inform the development of new rational therapeutics for pediatric astrocytomas and other cancers with MYB transcription factor alterations.